PURIFICATION AND CHARACTERIZATION OF THE LIPASE FROM SERRATIA-MARCESCENS SR41-8000 RESPONSIBLE FOR ASYMMETRIC HYDROLYSIS OF 3-PHENYLGLYCIDIC ACID-ESTERS

A new lipase which enantioselectively hydrolyzes (+/-)-trans-3-(4-meth oxyphenyl)glycidic acid methyl ester [(+/-)-MPGM], a key intermediate in the synthesis of diltiazem hydrochloride, was purified from the cul ture supernatant of Serratia marcescens Sr41 8000. The apparent kineti c constants (K(m), V(max)) for hydrolysis of (2S,3R)-MPGM [(+)-MPGM] w ere 350 mM and 1.7 x 10(-3) mol/min/mg protein in a toluene-water (1 : 1) emulsion system. The lipase did not attack (2R,3S)-MPGM [(-)-MPGM] , and (-)-MPGM acted as a competitive inhibitor. The molecular mass wa s estimated to be 62,000+/-2,000 from SDS-PAGE. The lipase preferentia lly hydrolyzed (2S,3R)-3-phenylglycidic acid esters, but did not hydro lyze cinnamic acid esters. The lipase released glycerol and fatty acid from olive oil, and the optimum pH and temperature for hydrolysis of olive oil were pH 8 and 45-degrees-C, respectively. The lipase was inh ibited by Co2+, Ni2+, Fe2+, Fe3+ and EDTA, and activated by Ca2+, Liand SDS. It was presumed that the lipase was a metalloenzyme containin g approximately one gram atom of calcium per molecular mass of 62,000. The lipase selectively hydrolyzed the 1,3 ester of triglycerides. Seq uencing of the N-terminal amino acids revealed that this lipase was di stinct from other known lipases.